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Trabalho de Tecnologia Genética: Diagnostico Molecular e Bioinformática = APS Aluno: Clebson Barbosa carvalho RA: 20623517 Professor: Daniele Gonçalves Castilho struções para a APS: A técnica de PCR (Polymerase Chain Reaction – Reação em Cadeia da Polimerase) é atualmente amplamente utilizada para o sequenciamento de genes e diagnóstico de doenças hereditárias, identificação de impressões digitais do material genético e detecção e diagnóstico de doenças infecciosas. Por ser um método direto de alta sensibilidade, a reação em cadeia de polimerase está sendo considerada uma grande aliada na detecção de doenças infecciosas. Sua versatilidade, que permite a aplicação em diversos materiais biológicos, aliada à rapidez de análise é outro diferencial da técnica (http://www.cjflash.com.br/cace/informe,biologia-molecular-porpcr,7.html). ATIVIDADE 1: 1) Assista os vídeos disponíveis nos links: PCR: https://pt.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcrelectrophoresis/v/the-polymerase-chain-reaction-pcr Eletroforese: https://pt.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcrelectrophoresis/v/gel-electrophoresis-dna ATIVIDADE 2: Leia os textos disponíveis nos links: Reação em cadeia da polimerase: https://pt.khanacademy.org/science/biology/biotech-dna-technology/dnasequencing-pcr- electrophoresis/a/polymerase-chain-reaction-pcr Eletroforese em gel: https://pt.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcrelectrophoresis/a/gel-electrophoresis Sequenciamento de DNA: https://pt.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencingpcr- electrophoresis/a/dna-sequencing ATIVIDADE 3: O aluno deverá copiar as questões abaixo, colar num documento de preferência, e responder qual a reposta correta. 1- Qual seria o fluxo em um laboratório de biologia molecular para se obter o resultado de um paciente que está realizando uma pesquisa de bactéria por PCR? X a) Extração de DNA --> PCR --> Eletroforese b) Eletroforese --> PCR --> Extração de DNA c) PCR --> Eletroforese --> Extração de DNA d) Eletroforese --> Extração de DNA --> PCR e) PCR --> Extração de DNA --> Eletroforese Resposta: a) 2- A reação da PCR consiste em amplificar um fragmento (região específica) de DNA milhões de vezes, para que isso ocorra precisamos preparar a reação previamente. Que reagentes são necessários para a montagem de uma reação de PCR? X a) água, buffer, MgCl, DNTPs, Taq polimerase, primers e DNA b) água, buffer, helicase, DNTPs, Taq polimerase, primers e DNA c) água, buffer, Mgcl, DNTPs, DNA polimerase, primers e DNA d) água, buffer, helicase, DNTPs, DNA polimerase, primers e DNA e) água, buffer, primase, DNTPs, Taq polimerase, primers e DNA Resposta: a) 3- Qual é o propósito para o qual a PCR é usado? a) Analisar a expressão gênica do DNA b) Extrair o DNA de células X c) Amplificar em fragmento específico de DNA d) Fazer todo o processo de replicação igual a célula e) Analisar o tRNA presentes nas células Resposta: c) 4- O que acontece com o DNA quando o termociclador atinge a temperatura de 90-95 ° C por 30 segundos no primeiro passo da PCR? a) Os primers se ligam na fica única de DNA b) A taq polimerase sintetiza as novas fitas do DNA, utilizando os nucleotídeos provenientes da DNTP c) Ocorre a desnaturação de proteínas X d) As fitas de DNA se separam e) Ocorre a renaturação da molécula de DNA Resposta: d) 5- As seguintes etapas constituem a reação em cadeia da polimerase (PCR), onde milhões de cópias do fragmento de DNA que se quer amplificar são gerados, I. Desnaturação do DNA genômico (94ºC, 30s). II. Anelamento dos iniciadores no DNA genômico (55-65ºC, 30s). III. Desnaturação do DNA genômico (94ºC, 5min). IV. Síntese do DNA pela polimerase (72ºC, 2-5 min). A sequência correta da reação apresentada está na opção: X a) I - II - III - IV b) III - II - IV- I c) II - I - IV- III d) IV - I -III-II e) III - I - II - IV Resposta: a) Desenhar Primers: SAP-PADG_0 0634 50D-PADG_0 7418 FORMAMIDASE- PADG_00315 Hypothetical Protein-PADG_ 04791 Glutamate cysteineligase- NM_001203879.2 Seqüência Temp. Melting 62 62 62 %GC 50 50 47,6 Tamanho do primer 20 22 21 Comprimento aplication 312 804 Seqüência SAP-PADG_0 0634 50D-PADG_0 7418 FORMAMIDASE- PADG_00315 ATGGCAACCCAAATCCAACAAAAGCTCCAAGACAGAAATGCCGGATATGCAAACACTTTCAATCAGAGTCATCTTGCCATTCCCCCTGTGGAGAAATATCTTGTTCGTATGTTAACCCATACCCCCTCTCCATTGCCCCTCTAGTAACTCTTCAACAGTGACCTGTATGGATGCCCGTATCGACCCCTCCTCCGCCTTTGGCATCTCCCTTGGAGATGCCCACGTAATCCGTAATGCCGGCGCCAGCGCCCGCGATGGCTTGCGCTCCATTGTCATCTCGCAGCAGCTGCTAGGAACCCGCGAGATCCTGCTCATCAAGCACACGGGCTGTGGCATGCTGACCTTCACCAACGAGCAGGCGCATGCTCTTGTTGAGAAGAAATTGGTGGAGAGTCACAGGCAGAATGGAACTGGCGCTGCGGATGGCGGGTGTAGGGTGCTCGAGGCGCTGAAGAGGGATATTCCGGATTTCCAGCCGTTTCCACTGCTT GAGGAAGCGGTTAGAGAGGATGTTAGGTGGCTGAGTGAGCAGGTGCTGGTTACGCCTGGGATTCCAATTTCCGGGTGGGTGTATGAGATTGAGACGGGGAAGGTGAGGAAAGTTGTGTAG Hypothetical Protein-PADG_ 04791 ATGTACATGATCATACGAAGAAACACGTTCACTGGTGTCCATGATCAAAATTTAATGTTTTACGTAAAAA GTACAGATGTTAACTGTTTAGTTTAA Glutamate cysteineligase- NM_001203879.2 ATTTTCGTTCATCAGAACCAAGATTTTTTTTGTCGACACAATCAAACCAAATAGATAATGATACTGTTTTTTATAGTAGTATATATATATTTCTTTTCCTATAGAGTATTTTATCTGAGGTCATGTGGTTGGTTAGTGACACACTTTTCATTATTTTACGTTCTAAATTAAATATCGTAAAGTTAAACCTCGTTTAAAGTATACTATAAAGAATAGAAATATCATTAATTCATTATACACACTATAAAGTTAACCTTTGTTTAAACTACTAATAAACTGGTTATTTAAAAAACTATTAGGGTTAAATTTGTGAGAAATGGAAAAGTAAGAGGCAAAAAACGAAAGAATCTTTT ATTACAATGATTATTAATTTTATCAACTTTCTTTTATAGACAGCTCAGCTCCACTGATCGGCTCATGCTCCACTGATCAACTTTCTTTCATTTTTTTATCAGTTTCTCATTTCTCTCTCAGTCGCATATAAATTTGAAAC GCCGATTCGTTTTTTGCTCTTTAAGCTTTCTTCTTGATTTCGACGGTGTCTCTCAATCTCCGTCAAGCTTG ACGAATTTCAGGTGTGATTTGCTTCTCTTCTTCTTCGCTTTCTGGACTTGTGGATTTTGGTTTTTTGATTG ATTTGTTCTGCTTTTTTTTTTTTGGTTGAATCTTCGCTTTTTCTCATGATTTTTTTTTTGGCTGTTGCATG ATTTTGGTTTTTGTTAGAGTCGAATTTTTTGCAGATTTTTGCTTTGATTTGATTTTTGTTGTTGTTGTTGTT TCTGGTTTTGGTGATACACGAATTTTGATTTGTTCTTCCAGATGATAGTTCAATTCTGTGGTAGCTGCTGA TTCTAATTATGTGATATATGTTATTAGAATTGAGAAAAATGCAGTTATTTGTGATTTCTCATAGCTTATG ATGAATTGTTATAATTGCAGGAGCTATATATACCATGGCGCTCTTGTCTCAAGCAGGAGGATCATACACT GTTGTTCCTTCTGGAGTTTGTTCAAAGGCTGGAACTAAAGCTGTTGTTTCGGGTGGCGTGAGGAATTTGG ATGTTTTGAGGATGAAAGAAGCTTTTTGGTAGCTCCTACTCTAGGAGTCTATCTACCAAATCAATGCTTCT CCATTCTGTTAAGAGGAGTAAGAGAGGGCATCAATTGATTGTTGCGGCAAGTCCTCCAACGGAAGAGGCT GTAGTTGCAACTGAGCCGTTGACGAGAGAGGATCTCATTGCCTATCTTGCCTCTGGATGCAAAACAAAGG ACAAATATAGGTACAGTTTGATGCTTATATGTTCTGTTAAAATGCTACTTAGTGTTTGTAATTGTGACAT ATTTCTTTGTTTGTGCTCTTTTACGGTGTTGGTATTGTTTGAATTCATCCAGATTGTTTGTCTATTGGAG AAATATTCTGTTTTTGCTAACTTAACATTGTGAGTTTTCAGAATAGGTACAGAACATGAGAAATTTGGTTT TGAGGTCAATACTTTGCGCCCTATGAAGTATGATCAAATAGCCGAGCTTCTTAATGGTATCGCTGAAAGA TTTGAATGGGAAAAAGTAATGGAAGGTGACAAGATCATTGGTCTGAAGCAGGTTAACATTCTCCTGTGTT TCATCTTTTGAGCCGTTTATTATTTATATACATTTTCAGCCTGTAGAACATGTTAGCTTCATATACAACA CAATGATCGGTATATTATAAGGTTTCAGTCTTTTTTTTTTTTTTTGCTGTAGATAACTCGTTCTGTCCTTT TGTTGACCAGGGAAAGCAAAGCATTTCACTTGAACCTGGGGGTCAGTTCGAGCTTAGTGGTGCACCTCTT GAGACTTTGCATCAAACTTGTGCTGAAGTCAATTCACATCTTTATCAGGTAATTTCAAAGATAATTATTG CCAATGCTCTCACCCTAAAACGTGACAAATAACCAACTATCTACGGATGTCACAGGTAAAAGCAGTTGCT GAGGAAATGGGAATTGGTTTCTTAGGAATTGGCTTCCAGCCCAAATGGCGTCGGGAGGATATACCCATCA TGCCAAAGGTATGACGCTTTCTCTATTCTTAAGATTGAAGAAAGTTAACATCTGTTTGTTTCTAATTCAG ATAATATTATCTGATTCTACATAGCCAACGTTCGGATTATTTCTTGGTGTTTGTGTTAAGTTTTCCTATG ACGAGTCAGGCAGTAAGATTTGACATAGTGTAATGTGTTCTGTGCAGGGGAGATACGACATTATGAGAAA CTACATGCCGAAAGTTGGTACCCTTGGTCTTGATATGATGCTCCGAACGTGTACTGTTCAGGTTAGATTA ATATTCTGACTTTGCGCCTTTTCATAATTTCCTTAAACTAAACTTTCCAGTTCTCATCAATAATCTTATT TTGAAACTTATAGAGAAACTGCATTACTTGCAATCTTGACAGGTTAATCTGGATTTTAGCTCAGAAGCTGAT ATGATCAGGAAGTTTCGTGCTGGTCTTGCTTTACAACCTGTGAGAAGCTCGATCTCTTTGTGGCATAACC AGAAACTTTTATTAACGATTTTGACTAACCATTGCCTCACTATTATCTAAATTCAACAGATAGCAACGGC TCTATTTGCGAATTCCCCTTTTACAGAAGGAAAGCCAAACGGATTTCTCAGCATGAGAAGGTATCTAGAT TCAGTTGTTATGATCCCCGATATCTTTTTGAATTAGTTTCCCTCCCACCTTATAACTCTGGTATGTTTTG ACAGCCACATATGGACAGACACTGACAAGGACCGCACAGGAATGCTACCATTTGTTTTCGATGACTCTTT TGGGTGAGTATCGATGTTTAATCTATGAAAAACAATTACATTACATTGGTTGGTTTGGTATGAGAAGCTTCTCAAAAACCAATGACTCTGTCTCGCATTCTTGATGAGTTATTGGAAAATTGATATGTGCTATTCTGTGG ATTTTCTTACATATGTTTGTGTTCAAGATAGAAAGATGCTTTGGATCTTTATCCTTCTCTTGAAACAGCA TGGCTAATCTTAATTTTATTGCTCTCTTTTGTTCTTATCTGGTATAGGTTTGAGCAGTATGTTGACTACG CACTCGATGTCCCTATGTACTTTGCCTACAGAAAGAACAAATACATCGACTGTACTGGAATGACATTTCG GGTCAAAATCTATCCTTAATCTTTTCTGTTAGGTTCTATAACTGTTTCATTGCAAGGAATTGGCAAATTG AAAACCATGGGATCTTCTTGAATTATATCTAATCTCATAATATTCTTCATGCTGCAGCAATTCTTGGCTG GAAAACTTCCCTGTCTCCCTGGTGAACTGCCTTCATATAATGATTGGGAAAACCATCTGACAACAATATT CCCAGAGGTTAATCATCTGCCTCAAGAGTCTTTCAATCAGCCTATGACTTTCATTTCCCTGTCTTATGTT GAAGTCTGACAACGTTCCATAACACTAGGTTCGGTTGAAGAGATACTTGGAGATGAGAGGTGCTGATGGA GGTCCCTGGAGGAGGCTGTGTGCCCTGCCAGCTTTCTGGGTAACTACATCTATTAGCACATATATATTTC GTGCTTAATGAGAATGTTTCAAAAAGTATTGACTGTTGTTTTGATTTTATCTACAAATTTCAGGTGGGTT TATTATATGATGATGATAGTCTCCAAGCTATCCTGGATCTGACAGCTGACTGGACTCCAGCAGAGAGAGA GATGCTAAGGAACAAAGTAATAACAAGACGCACCACATTTACATTTTCTTATTCCACAGTTAGAAAACAT CTTGTAATAAACTAATACTTGATTTCCAACAGGTCCCAGTTACTGGCTTAAAGACTCCTTTTAGGGATGG TTTGTTAAAGCATGTCGCTGAAGATGTCCTGAAACTCGCAAAGGTGTGTTTGTATGAACCCTCTTAGAAA CTTCGAGCCAGCTTTCTATCCCGTCTGGTCCAGCTTCTAACTATTGGACTTATTTTCTCTTTGTTTCTCA GGATGGTTTAGAGCGCAGAGGCTACAAGGAAGCCGGTTTCTTGAACGCAGTCGATGAAGTGGTCAGAACA GGTAAATGAAGTTTCCTAATGCATAATTGAATTCCACACCGACATTTTGTTTGATAACCTGGTGGTGAAA TTTGGTTTTTGAAATGACAGGAGTTACGCCTGCGGAGAAGCTCTTGGAGATGTACAATGGAGAATGGGGAC AAAGCGTAGATCCCGTGTTCGAAGAGCTGCTGTACTAAGAAAATGGGACGTGAACAAAAGGTGTCTATAA ACCTTTGGGTGTGAGTTTATGCTATCTGAAGAATTCGAGTCTCCGGAATAAGGATTTTTTTTTTTTTGTTGT AATCGGATTTTAAAAACTGATTTTGTTTTAGAAATTCGAAGCATTGAAAAGCAGAAGAAAAATTGTATGT ACTAAACGATTTCGGTGTGGGAAATCGTTTGGGAGGGTGTGTTTGGATCTTGAATAAATTACCCATTTTT CTTGTCACAAATTTTGTCTACATTTAACGAAATAACTCAAAACTGATTTCAAGGCAAAGGCGACGGATGGG ATAATCTAGGCTGTGTAGTGGAAAGTTTTTGATGTTTAGTAATTTGGCAATGTATGTTTTTTTTCTAAATGG GAGAGCATCTTCGATGTAGACATTTACGAGCTTTTCCTTTAATAGGTTTTTGGCTAGTGAAGAACACTGCT TTCATAATTGAATCCACCAGACTCCGCGTAAACTAAACTGATTACAACACGATGATCCTGGCCGTCGGTA ATTATCGTAAAACAAGAAGATAGTGGCACTTAGGTGACAGGATAATATGTAAACCCTACCAAGGCGTCCC TACAAAATGATGAGTGATGACTGATGAGAGTGAGAAAAACCGAGTCTCCACAGTCCACACACTGCCTTAC CTAATTAATATACCCACCTTTCATTCATGTATGTTACGTCAACGTTTTTATGTCCGTTAAAACTGTAATC TACATAAGAGATCATAATTTTTAAAATCTCCATGTATTTCGTTGATACTTTAATTTGGCACGTTTCTAGT TTTTGAGATCAAATGTTTTTAATCTGCTTCTAGTCTTATTTTTCCTAAACCTTAAACTGTAAACCAATTG CTTTTTCACATAACCTTGCTTGTTTTCAATTTTAAAATTTAAGTCCAATATTTGAAACAACCTAACAAAT GTCGTCTATGGCAACCAAAAAAAAATAAAAAATCAAAACAATTTGCTAAAAAGGAAATTGATTAAAATTG ATTTCTTTCATTTAAATTGGAATTTAATATATTAGGTAAACATGTAAACGCAATCGCAATTTTTATGAGA GACTTCGGGCCGGTTCGTTTAAGTGTCGCTGCGACTATAACTAGCGACTGGCTTTAAAAACGACTGAGTT TTGTTTGTTTTTTATTGTTTTTTTTTTAAGTTCGAATATGTCGTATAGATCTGTAAAATAATTCAAATGTG GCTTTGTCCCATAGATTGGTTTTGATTATGCAATCATCTATCACAACAGTGGAACATTATGCAATCATTT ATTGTCTATCAAATTAATTATGGTTATGA
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